Defining the Catalytic Mechanism of the HDV Ribozyme. The hepatitis delta virus (HDV) is a human pathogen that causes severe acute and chronic liver disease. The viral genome contains the sequence for a ribozyme that is essential for replication. Although our understanding of ribozyme catalysis has advanced enormously in the two and half decades since their discovery, their mechanisms of catalysis remain largely undefined. Although crystal structures for many of these ribozymes have been solved, significant discrepancies between the structural data and functional experiments remain with many ribozyme systems including the HDV ribozyme. Additionally transition state structure for most ribozymes remains largely undetermined. In this proposal we will employ powerful chemical mutagenesis approaches together with atomic mutation cycles to reveal atomic connections in the molecule that are critical for function, including the connections between the ribozyme, the catalytic groups, and the reaction transition state. Concurrently, we will apply newly developed state of the art techniques in kinetic isotope effect (KIE) analysis (in collaboration with Michael Harris at Case Western Reserve University) to characterize the reaction pathway and obtain a measure of the bonding changes that occur during catalysis. We will then combine atomic mutagenesis with KIE measurements to determine how functional interactions in the ribozyme influence bonding in the transition state. This combination of approaches is unprecedented in RNA enzymology and promises to yield penetrating new insights into the catalytic mechanism of this RNA. Overall this work will establish an experimental and conceptual paradigm for studying other RNA enzymes and nucleotidyl transferases and the implications for biological catalysis will be far-reaching. PUBLIC HEALTH RELEVANCE: HDV is a human pathogen causing severe acute and chronic liver disease in humans. The viral genome contains a ribozyme (HDV ribozyme) required for viral replication, and the human genome contains a similar sequence of unknown function. Understanding the ribozyme's mechanism of action promises to shed light on fundamental aspects of RNA catalysis and reveal critical features of viral pathogenicity, which may lead to new treatments for hepatitis.